Project Summary/Abstract Enterovirus (EV) infections of the central nervous system (CNS) are a significant cause of morbidity and mortality worldwide. Although polioviruses are among the most well known neurotropic EVs, many non-polio EVs also cause CNS disease. In 2014, the United States experienced an unprecedented respiratory disease epidemic of a non-polio EV known as EV-D68. This outbreak was associated with a dramatic rise in the number of cases of acute flaccid myelitis (AFM), a paralytic condition with striking resemblance to poliomyelitis. There are currently no approved treatments for AFM. In addition, little is understood about why EV-D68 has only been recently associated with AFM or about the pathogenesis of AFM. A key barrier to understanding the pathogenesis of EV-D68 has been a lack of models of EV-D68 CNS infection. We have overcome this limitation by developing an in vivo model of EV-D68 CNS infection that shares many features with human AFM. In this model, EV-D68 infects motor neurons resulting in limb paralysis in mice. The viral infection can be quantified by standard viral titer assays (TCID50) and tracked in motor neurons by immunohistochemistry. In addition, our preliminary studies have shown that EV-D68 can grow in dissociated mouse motor neuron cultures. The objective of this proposal is to use these in vivo and in vitro models of EV-D68 CNS infection to improve our understanding of the viral genetic determinants and pathogenic mechanisms involved in both EV CNS infection and AFM. Aim 1 will investigate viral genetic determinants of neurovirulence and neuroinvasiveness using sequencing and molecular cloning techniques, combined with testing in the mouse model. This aim will allow for understanding of mechanism of infection based on regions of the virus important for neurovirulence and neuroinvasiveness. Aim 2 will investigate mechanisms of neuroinvasiness and spread using a microfluidic approach with cultured human induced pluripotent stem cell motor neurons and cortical neurons. Successful completion will likely provide fundamental knowledge into EV-D68 CNS disease pathogenesis and has the potential to identify novel therapeutic targets for treating EV CNS disease.